Recent developments in the pharmaceutical industry and in combinatorial chemistry have exponentially increased the number of potentially useful compounds, each of which must be characterized in order to identify their active components and/or establish processes for their synthesis. To more quickly analyze these compounds, researchers have sought to automate analytical processes and to implement analytical processes in parallel. Commonly employed analytical processes include chemical or biochemical separations such as chromatographic, electrophoretic, electrochromatographic, immunoaffinity, gel filtration, and density gradient separations.
One particularly useful analytical process is chromatography, which encompasses a number of methods that are used for separating ions or molecules that are dissolved in or otherwise mixed into a solvent. Liquid chromatography (“LC”) is a physical method of separation wherein a liquid “mobile phase” (typically consisting of one or more solvents) carries a sample containing multiple constituents or species through a separation medium or “stationary phase.” Stationary phase material typically includes a liquid-permeable medium such as packed granules (particulate material) or a microporous matrix (e.g., porous monolith) disposed within a tube or similar boundary. The resulting structure including the packed material or matrix contained within the tube is commonly referred to as a “separation column.” In the interest of obtaining greater separation efficiency, so-called “high performance liquid chromatography” (“HPLC”) methods utilizing high operating pressures are commonly used.
In the operation of a separation column, sample constituents borne by mobile phase migrate according to interactions with the stationary phase, and the flow of these sample constituents are retarded to varying degrees. Individual constituents may reside for some time in the stationary phase (where their velocity is essentially zero) until conditions (e.g., a change in solvent concentration) permit a constituent to emerge from the column with the mobile phase. The time a particular constituent spends in the stationary phase relative to the fraction of time it spends in the mobile phase will determine its velocity through the column.
Early parallel LC systems that coupled multiple conventional tubular columns to common fluid supply and/or control systems provided only marginal benefits in terms of scalability and reduced cost per separation. Recent advances in microfluidic technology have allowed fabrication of microfluidic multi-column HPLC devices that permit simultaneous (parallel) separation of multiple samples while consuming very small quantities of valuable samples and solvents and generating much smaller volumes of liquid waste. Examples of such devices are disclosed in commonly assigned U.S. patent application Ser. No. 10/366,985 filed Feb. 13, 2003 (now publicly available as U.S. Patent Application Publication no. 2003/0150806), which is hereby incorporated by reference. These microfluidic devices require far fewer parts per column than conventional HPLC columns, and may be rapidly connected to an associated HPLC system without the use of threaded fittings, such as by using flat compression-type interfaces either with or without associated gaskets. A further benefit of microfluidic parallel HPLC devices is that their relatively low cost and ease of connection permits them to be disposed of after a single or only a small number of uses, thus eliminating or dramatically reducing the potential for sample carryover from one separation run to the next.
Many desirable analyses utilizing HPLC require some preparation of the sample prior to the analysis. Examples of common sample preparation processes include, without limitation, metering, pH adjustment, dilution, addition of standards, solvent extraction, addition of reagents, reconstitution, size exclusion filtration, chemical affinity filtration (including solid phase extraction), centrifugal separation, molecular weight cut-off membrane filtration, dialysis, liquid-phase extraction, protein precipitation, etc. Sample preparation may also include fluid processes such as qualitative or quantitative tests including various types of assays.
These sample preparation steps often may be time consuming, labor intensive, and create the opportunity for error to be introduced (e.g., by incorrect measurement of quantities or concentrations). It is relatively simple to prepare samples for analysis in single-column HPLC systems using conventional laboratory techniques and equipment. Extending such preparations to multi-column (e.g., parallel) HPLC systems, however, is significantly more challenging due to the sheer volume of samples that must be prepared simultaneously to take advantage of high throughput capacity of microfluidic parallel HPLC devices. For example, if multi-column HPLC system can process twenty-four samples in a single run, twenty-four samples must be prepared prior to initiating the run. If samples are processed serially, the cycle time associated with the sample preparation process could eliminate the time savings provided by the parallel analysis. In addition, a first set of prepared samples could degrade, evaporate, or otherwise be damaged while awaiting preparation of the remaining samples for a particular run.
Moreover, traditional sample preparation processes and devices are designed for conventional scale (i.e., non-microfluidic) laboratory devices. Thus, while microfluidic parallel HPLC devices or other synthesis and/or analytical systems may require only very small sample volumes, conventional sample preparation processes and devices typically create large volumes of sample, much of which would be discarded as waste if such prepared samples were supplied to microfluidic systems. Because samples may often include scarce and costly chemicals, such waste would be undesirable. Additionally, samples often contain hazardous or toxic materials with their attendant safety and disposal concerns. In addition, traditional sample preparation devices may be complex and expensive to manufacture. As a consequence, economical operation may require devices to be cleaned and re-used, further increasing the procedures required to cycle the instruments and related equipment.
Thus, it would be desirable to provide microfluidic devices for preparing samples for analysis in microfluidic parallel HPLC devices. It would also be desirable to provide microfluidic devices for preparing substantially all samples to be used in a particular analysis operation simultaneously to minimize sample degradation or other detrimental effects of prolonged delays prior to the (e.g., serial) analysis of individual samples. If would further be desirable to provide microfluidic devices capable of performing sample preparation processes while minimizing the consumption of samples and reagents. It would also be desirable to provide microfluidic devices for preparing multiple sample sets simultaneously to minimize cycle times between analysis runs. It would further be desirable to provide a system capable of preparing a set of samples while another set of (previously prepared) samples is being analyzed to minimize delays between analytical runs. It would also be desirable to provide microfluidic sample preparation devices that are easily fabricated and disposable.